Disc regeneration

A GLIMPSE OF THE NOVEL INTERVENTIONAL APPROACHES

Babak Ashrafnejad MDPain FellowshipAnesthesiologist

Low back pain is a disorder that affects a considerableproportion of the population

About 60–80% of all people suffer from back pain atsome time during their life

Degeneration of the intervertebral disc (IVD) and discherniation are two distinct but related causes of lowback pain and radicular pain, respectively

At least 40% of patients with chronic low back painshowed characteristics of intervertebral discdegeneration (IVDD)

IVDD is an aberrant, cell-mediated response toprogressive structural failure

ANATOMY

Fibrocartilaginous tissues that allow motionbetween the vertebral bodies

They transmit load and absorb the shocks

Each IVD is composed of three distinct butconnected structures:

The Endplate

The NP

The AF

Endplates consist of hyaline cartilage and occupy theinferior and superior interfaces between theintervertebral disc

Collagen is greatest at the periphery of the endplates, while the centre contains most of the proteoglycansand water

CollagenProteoglican

+Water

They have approximately 1-mm-thick horizontal layer of hyaline cartilage

Early in life : the endplates are highly vascularized

First year : The degree of vascularity wanes dramatically

Third decade : No blood vessels present

Encases the NP and prevents the it from herniating

The AF is composed of :

Water (65–90% of its weight)

Collagen type I and II fibres (60% of dry weight)

Proteoglycans and other proteins (10–20% of dryweight)

The AF is composed of 15–25 loosely connected concentric rings of highly organized collagen fibres(lamellae)

Has a cell density of 9×106/cm3

Only at the outer layer, there is sensory nerves

Nutrition of the IVD is based on diffusion of nutrients through the subchondral bone and the endplates to the disc

Much less dense and nearly the consistency ofa gel.

Gel contains :

1- Type II collagen and elastin. These hold the gel-like area

2-Proteoglycan molecules that have hydrophilicchondroitin and keratin sulfate attached tothem.

3- Water > 90%

Water Molecules

proteoglycan

Collagen fibers

Is the disc framework structure

Composed of collagen and aggrican

Makes disc components more strenght (anchor)

MatrixChodrocytes

Collagenfibers

1, intervertebralDisc2, posterior longitudinal ligament 3, spinal nerveroot4, vertebral body

5, segmental artery 6, interosseous arteries7, dorsal root ganglion with accompanying blood vessel 8,descending branch of the sino-vertebral nerve 9, ascending branch of the sino-vertebral nerve 10, aorta

1, intervertebral disc

2, recurrent sino-vertebral nerve3, spinal nerve4, posterior branch of spinal nerve5, thecal sac with spinal roots6, paraspinal muscle

DISC DEGENERATION

Body weight

Lifting strength

Ageing

Occupational risks, such as exposure to vibrations

Smoking and atherosclerosis causing decrease in nutrient supply

Tissue weakening, which primarily occurs due to inherited genetic factors

Nutritional compromise

(Adams and Roughley, 2006)

catabolic processes exceeding anabolic ones

morphological changes, cell transformations and degeneration

loss of proteoglycans and a decrease in water content of the NP

Degeneration of the endplates impairs transport of nutrients into the disc and results in the accumulation of waste products such as

lactic acid, which reduce the pH

reduce the number of cells / reduction of extracellular matrix (ECM) synthesis

Dehydration of the NP

Reduced shock absorbance capacity

Loads will be transferred to the AF

Ruptures or cracks of the AF

Fibrosis

Narrowing of the disc space

Diffuse bulging of the annulus beyond the disc space

Extensive fissuring

Mucinous degeneration of the annulus

Defects and sclerosis of the endplates

Osteophytes at the vertebral apophyses

DISC REGENERATION

Anticatabolic agents

Growth factors

Gene Therapy

Cellular componentsNp cell transplantation Stem cell Therapy

Scaffolds

ANTICATABOLICS

The MMP (Matrix-Metalloproteinase) enzymefamily are responsible for the degradation of :Collagen

Aggrecan

Versican

Within the matrix, MMP activity is normallyinhibited by TIMPs (Tissue Inhibitor ofMetolloproteinase)

MMPs & TIMPs are balanced in normal discs

When imbalance Degeneration

Four TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) have been identified in discs.

Application of TIMPs for treatment of DDD through gene therapy or direct protein application (is still in an early phase of development)

ABSTRACT Cells from degenerated intervertebral discs were transduced with an adenoviral vector delivering cDNA of the catabolic

inhibitor, TIMP-1, and alterations in the measured proteoglycan were assessed.To assess the potential of TIMP-1 to favorably modify the proteoglycan content of degenerated intervertebral disc cells.Gene therapy with anabolic factors has resulted in increased proteoglycan synthesis in intervertebral disc cells. Biochemical analysis of degenerated discs has revealed elevated levels of the catabolic enzymes, matrix metalloproteinase, suggesting an intimate role of these factors in the degenerative process. The use of TIMP-1, an endogenous inhibitor of matrix metalloproteinase, via gene therapy may provide an additional method to alter the degenerative processes occurring in the intervertebral disc.Degenerated intervertebral disc were isolated from eight patients undergoing elective surgical procedures. Cells were cultured in monolayer and transduced with different concentrations of either an adenoviral-tissue inhibitor of metalloproteinase-1 (Ad-TIMP-1) or adenoviral-bone morphogenic protein-2 (Ad-BMP-2) construct. Cells were cultured in a three-dimensional pellet and proteoglycan synthesis was assessed via 35S-sulfur incorporation.Gene delivery of TIMP-1 and BMP-2 increased measured proteoglycan synthesis at each concentration assessed. IVD cells treated with Ad-TIMP-1 demonstrated an optimal response at a multiplicity of infection (MOI) of 100. Cells treated with Ad-BMP-2 demonstrated a progressive increase in proteoglycan synthesis with increasing viral concentrations.

Successful delivery of the anticatabolic gene, TIMP-1, results in increased measured proteoglycan in cultured degenerated disc cells. This finding supports catabolic inhibition as a promising avenue of research for the treatment of degenerative disc disease via gene therapy.

Wallach C J .et al. Spine , Oct 2003

IL-1 and TNF have been shown to mediate the inflammatory cascade in discs and

contrariwise,

anticatabolic agents, such as inhibitors of IL-1 & TNF have been shown to be

chondro-protective

Disc degeneration diagnosis based on history, physical exam and Imaging studies.

Positive discography ( VAS>6 at <50psi above opening pressure)

Intradiscal etanercept injection

Sainoh et al. 2014 Intradiscal etanercept inj. (RCT) (10 mg)Results : positive response

Cohen et al. 2007. Intradiscal etanercept inj. (RCT) (0.1-1.5 mg)

Results : no positive response

Cohen et al. 2009. Transforaminal epidural etanerceptinjection (RCT) (2-6 mg)

Results : is superior to systemic application in sciatica pain relief

GROWTH FACTORS

Growth factors bind to cell membranes(receptors)

activation of an intercellular signaling cascade

exert biological effects, such as :

1- Stimulates cell proliferation, differentiation, migration

2- Regulates matrix production and repair

Autologous Platelet-Rich-Plasma (PRP)

Platelet derived transforming growth factor-13 (TGF-I3)

Epidermal growth factor (EGF)

Osteogenic protein-l (OP-l) also known as bone morphogenic protein-7 (BMP)

LIM mineralization Protein 1 (LMP1)

Link N

SOX-9

EGF : increases in matrix synthesis and cell proliferation

TGF-I3I : matrix synthesis alone (TGF-I3I)‘

Increases in matrix synthesis with TGF-131 is dramatic.

Osteogenic protein-l (OP-l) into rabbits resulted in mild increases (12%-15%) in disc height up to 8 weeks following injection

BMPs are members of the TGF super family

Stimulates cells to express a more chondrocyticphenotype

Increase cell proliferation, and collagen Type II synthesis, aggrecan

Gene Therapy

transfer of ‘‘the gene of interest “ (RNA,DNA)

into the target cells using a so-called vector (Virus, main cells)

produce the desired gene products (RNAs or proteins) in a continuous fashion

Regeneration

1- Virus Vectors (Adenovirus/ Retrovirus)2- Non viral Vectors (direct gene transfer)

Vectors

Gens

Target Cell

Continuous Production

RNA

DNA

Continuous Biological Effects

Vector binds to cell memebrane

DNA injected to vector

Vector is packaged in vesicle

Vesicle breaks down releasing vector

Cell makes protein using new gen

Vector (Adenovirus)

Vector injects new gen into nucleous

Microbubbles Ultrasound Gene Therapy

NUCLEUS PULPOSIS CELL TRANSPLANTATION

Autologous reinsertion of the NP from normal disc to degenerated one has been shown to :

Delay degeneration of the disc, including the AF, NP, and endplate

Restoration of disc height

Retard disc degeneration in vivo

Increases production of collagen 2

No host-versus-graft response

Reimplantation of disc cells from discectomyspecimens in a nonrandomized series of human patients demonstrated MRI scan improvements consistent with increased

proteoglycan matrix within the NP and relief of symptoms

STEM CELL THERAPY

MSCs and bone marrow stem cells (BMSC) are the main stem cells

Readily available source of autologous cells, with minimal donor-associated complications

Adipose tissues are good source of stem cells(ADSC)

MSCs differentiated into cells expressing a chondrocyte-like phenotype (NP cells)

with increased production of :

matrix components, such as

Aggrecan

Collagen Type II

There are two main strategies for acquisition of these desired somatic stem cells:

Embryonic stem cells (ESCs)

from the fat or bone marrow

Stem Cell Therapy Procedure no general anesthetics are used.

The out-patient procedure takes about 3-4 hours:

1. Collection of adipose tissue by tickle lipo, centrifugal separation of stem cells

2. Collection of bone marrow aspirate from the posterior iliac crest, centrifugal separation of stem cells

4. Injection of the cells into the affected site under x-ray fluoroscopic guidance

to your disc

Yoshikawa et al , 2010

percutaneously grafted MSCs into degenerated IVDs in two women aged 67 and 70 years.

After two years, both individuals had alleviation of symptoms and radiographic changes

Haufe et al., 2006

intradiscal injection of hematopoietic stem cells into ten patients

none of these individuals had any relief of symptoms

SCAFFOLDS

The purpose of a cellular scaffold is to provide an optimal microenvironment

for

cellular migration and proliferation

that allows the cells to maintain the appropriate phenotype

Collagen is a physiological biomolecularscaffold (collagen gels)

hyaluronan acts as an anchor for aggrecanretention

Chitosan-based polymers a soluble polymer atroom temperature, and induced to gel at bodytemperature

Polyglycolic acid, polylactic acid

copolymers

Bioglass

Poly ε-caprolactone (PCL)

Polyurethanes

Silk (natural biopolymer)

BIOMATERIALS

Two broad categories :

polymers that are preformed

polymers that are formed in situ

Injectable

Require minimally invasive procedure

Radiopaque

The mechanical properties of polymers: Viscoelasticity

Toughness

Permeability

Conformable structure

Flowable materials may be injected via a smallincision, allowing minimally invasive access tothe disc space

Derived from silk and elastin

Mimics the protein content, water content, pH of the natural nucleus pulposus

Indications : early stages of Degenerative Disc Disease (DDD) and as an adjunct to microdiscectomy